Joshua B. Wechsler

Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome.

Children with Down syndrome have a 10–20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein, the genetic alterations that promote Down syndrome–related AMKL (DS-AMKL) have remained elusive. Here we show that leukemic cells from every individual with DS-AMKL that we examined contain mutations in GATA1, encoding the essential hematopoietic transcription factor GATA1 (GATA binding protein 1 or globin transcription factor 1). Each mutation results in the introduction of a premature stop codon in the gene sequence that encodes the amino-terminal activation domain. These mutations prevent synthesis of full-length GATA1, but not synthesis of a shorter variant that is initiated downstream. We show that the shorter GATA1 protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor Friend of GATA1 (FOG1; encoded by ZFPM1) to the same extent as does full-length GATA1, but has a reduced transactivation potential. Although some reports suggest that the activation domain is dispensable in cell-culture models of hematopoiesis, one study has shown that it is required for normal development in vivo. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

Post-Infectious Functional Gastrointestinal Disorders in Children

OBJECTIVE To investigate the development of functional gastrointestinal disorders (FGIDs) after an episode of acute bacterial gastroenteritis (AGE) in children. STUDY DESIGN A cohort study of children 3 to 19 years old with a positive result on a bacterial stool culture. 44 patients in each arm (unidirectional alpha of 0.05, power of 0.80). Children presenting at two pediatric hospitals (United States and Italy) for AGE who tested positive for bacteria on stool culture (2001-2005) were contacted at least 6 months after the episode. Exposed children were matched with control subjects of similar age and sex consulting to the same hospitals for trauma or well-child visit within 4 weeks of the index case. Symptoms were evaluated with a validated questionnaire for FGIDs assessing pain, diarrhea, and disability. RESULTS 88 patients (46 boys; mean age, 8.1 years; age range, 3-19 years) were recruited. Bacteria included Salmonella (54%), Campylobacter (32%), and Shigella (14%). 36% of exposed patients and 11% of control subjects complained of abdominal pain (P < .01). 87% had irritable bowel syndrome and 24% had dyspepsia. 56% reported onset of pain following the AGE. CONCLUSION There is a significant increase in cases of FGIDs after bacterial infections in children.

TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism.

Histamine is a bioactive amine that exerts immunomodulatory functions, including many allergic symptoms. It is preformed and stored in mast cells and basophils but recent evidence suggests that other cell types produce histamine in an inducible fashion. During infection, it has been suggested that neutrophils may produce histamine. We also observed that histamine is released in a neutrophil-mediated LPS-induced model of acute lung injury. Therefore, we sought to examine whether innate signals promote histamine production by neutrophils. Bone marrow-derived neutrophils stimulated with a range of TLR agonists secreted histamine in response to LPS or R837, suggesting TLR4 or TLR7 are important. LPS-driven histamine was enhanced by coculture with GM-CSF and led to a transient release of histamine that peaked at 8h post stimulation. This was dependent upon de novo synthesis of histamine, since cells derived from histidine decarboxylase (HDC) deficient mice were unable to produce histamine but did generate reactive oxygen species upon stimulation. Using pharmacological inhibitors, we show that histamine production requires PI3 kinase, which has been shown to regulate other neutrophil functions, including activation and selective granule release. However, unlike mast cells, HDC deficiency did not alter the granule structure of neutrophils, suggesting that histamine does not participate in granule integrity in these cells. Consequently, our findings establish that neutrophils generate histamine in response to a select panel of innate immune triggers and that this might contribute to acute lung injury responses.

Anaphylactic responses to histamine in mice utilize both histamine receptors 1 and 2

Anaphylaxis is a severe, potentially life‐threatening reaction that can occur in response to common triggers, including food allergens (e.g., peanut), insect stings, and several medications. Activation of mast cells and basophils to release preformed mediators, such as histamine, is thought to be an important process that underlies reactions. Histamine can exert effects through four different receptors, termed H1R–H4R. Despite clinical use of both H1R and H2R blockers in the therapy for acute allergic reactions, there is little mechanistic evidence to support the necessity for blocking H2R, a receptor best characterized for its role in stomach acid production.

Tetraspanin CD151 Is a Negative Regulator of FcεRI-Mediated Mast Cell Activation

Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, yet the mechanisms controlling mast cell activation are not well understood. Members of the tetraspanin family are recently emerging as modulators of FcεRI-mediated mast cell activation; however, mechanistic understanding of their function is currently lacking. The tetraspanin CD151 is a poorly understood member of this family and is specifically induced on mouse and human mast cells upon FcεRI aggregation but its functional effects are unknown. In this study, we show that CD151 deficiency significantly exacerbates the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Ex vivo, FcεRI stimulation of bone marrow–derived mast cells from CD151−/− mice resulted in significantly enhanced expression of proinflammatory cytokines IL-4, IL-13, and TNF-α compared with wild-type controls. However, FcεRI-induced mast cell degranulation was unaffected. At the molecular signaling level, CD151 selectively regulated IgE-induced activation of ERK1/2 and PI3K, associated with cytokine production, but had no effect on the phospholipase Cγ1 signaling, associated with degranulation. Collectively, our data indicate that CD151 exerts negative regulation over IgE-induced late phase responses and cytokine production in mast cells.

114 A Multicenter Study Assessing the Clinical, Endoscopic and Histologic Response to Four Food Elimination Diet for the Treatment of Eosinophilic Esophagitis

G A A b st ra ct s calculated at baseline and at week 12. Proximal and distal esophageal scores, total scores (summation of proximal and distal), and subscores for individual component of EREFS (edema, rings, exudates, furrows, stricture) were prospectively recorded. Baseline and followup EREFS scores were compared, and post-treatment eosinophil counts and EREFS scores were correlated. Data analysis was performed on the intent-to-treat population. Results: A total of 93 subjects were randomized from 25 centers, and 87 were included in the final analysis. 97% of subjects had endoscopic features identified at baseline. The OBS (n= 49) and placebo (n=38) groups did not differ in baseline demographic and endoscopic characteristics. EREFS scores significantly improved after treatment in both proximal (3.4 to 1.5; p<0.0001) and distal esophagus (4.3 to 2.4; p<0.0001) with OBS but not placebo (proximal 3.3 to 3.4; distal 3.6 to 3.9). Features of edema, rings, exudates and furrows showed significant improvement with OBS but not placebo (Figure). Strictures did not significantly change following OBS or placebo although subjects with high grade strictures were excluded from trial entry. Proximal, distal and total EREFS correlated with peak eosinophil counts after treatment (R: 0.35, p<0.0001). Conclusions: (1) This is the first study to utilize a validated endoscopic scoring instrument in a randomized controlled trial of medical therapy for EoE. (2) Significant benefit was demonstrated in the inflammatory (edema, exudates, furrows), ring, and total EREFS scores. (3) Significant correlation was demonstrated between EREFS and peak eosinophil counts. (4) Endoscopic outcomes may be important endpoints of EoE clinical trials that complement symptom and histologic assessments. Written on behalf of the MPI-101-06 Investigators.

IgE‐associated food allergy alters the presentation of paediatric eosinophilic esophagitis

Links between food allergens and eosinophilic esophagitis (EoE) have been established, but the interplay between EoE‐ and IgE‐associated immediate hypersensitivity to foods remains unclear.

Utility of Gastric and Duodenal Biopsies during Follow-up Endoscopy in Children with Eosinophilic Esophagitis.

Objectives: Eosinophilic esophagitis (EoE) is a chronic antigen-mediated immune disorder of the esophagus. Consensus guidelines recommend obtaining esophageal, gastric, and duodenal biopsies at diagnostic endoscopy when EoE is suspected. The utility of repeated gastric and duodenal biopsies during follow-up endoscopy in patients previously diagnosed with EoE is not established. The aim of the present study was to explore the role of gastric and duodenal biopsies in children with an established diagnosis of EoE undergoing repeat endoscopy to assess histological response to treatment. Methods: Retrospective chart review of children diagnosed with EoE at a tertiary care center was conducted. A total of 160 patients with EoE with demographic clinical, endoscopic, and histological data at diagnosis and follow-up endoscopy were included. The frequency of gastric and duodenal biopsies at follow-up endoscopy with abnormal histology and their correlation to endoscopic findings was determined. Results: At follow-up endoscopy, 83% (132/160) of patients had gastric and 74% (118/160) had duodenal biopsies. Histology was normal in 81% of gastric and 92% of duodenal biopsies. The most frequent gastric abnormalities were chemical and inactive chronic gastritis. The most frequent duodenal abnormality was villous blunting with increased intraepithelial lymphocytes. Two patients with normal gastric and duodenal histology progressed to eosinophilic gastroenteritis at follow-up endoscopy. Conclusions: Gastric and duodenal biopsies obtained in EoE patients during follow-up endoscopy show pathology in a minority of patients, increase costs, and may add potential risk of adverse events. Large multicenter, prospective studies of endoscopic practice during follow-up of EoE are warranted to provide evidence supporting best practices.

Eosinophilic Esophagitis Reference Score Accurately Identifies Disease Activity and Treatment Effects in Children

Background & Aims: The endoscopic reference score (EREFS) is used to determine severity of 5 endoscopic findings: edema, rings, exudates, furrows, and strictures. Little is known about the relationship between EREFSs and histologic markers of disease activity in children with eosinophilic esophagitis (EoE). We aimed to determine whether the EREFS can be used to identify children with EoE and how it changes with treatment. Methods: We performed a prospective study of consecutive children (ages 2–17 years) undergoing diagnostic or post‐treatment endoscopy scored real‐time with EREFS from December 2012 through 2016. Findings from 192 diagnostic endoscopies and 229 post‐treatment endoscopies were evaluated, from 371 children. Incident EoE cases were diagnosed based on 2011 consensus guidelines. Patients were treated with either elimination diet or topical steroids. Subjects who underwent endoscopy for symptoms of esophageal dysfunction but had normal esophageal findings from histology analysis were used as controls. EREFS and receiver operating characteristic curves were determined for incident EoE cases (n = 77) vs controls (n = 115), patients with active EoE (n = 101) vs inactive EoE after treatment (n = 128), and paired pre‐ and post‐treatment cases of EoE (n = 85). Component and composite scores were correlated with eosinophilia. Results: Visual detection of more than 1 esophageal abnormality during the diagnostic endoscopy identified children with EoE with 89.6% sensitivity and 87.9% specificity. EREFS correlated with peak level of eosinophilia (P < .001) at all esophageal levels. Children who responded to therapy had mean EREFSs of 0.5 compared to 2.4 in non‐responders. In comparing pre‐treatment vs post‐treatment data from 85 patients, we found a significant reduction in the composite EREFS (from 2.4 to 0.7) (P < .001) among patients who responded to treatment; 92% of responders had a reduced EREFSs after treatment. EREFSs identified children with EoE with an area under the curve value (AUC) of 0.93. EREFSs identified children with active EoE following treatment with an AUC of 0.81 before treatment and an AUC of 0.79 after treatment. Conclusions: In a prospective study of children undergoing diagnostic or post‐treatment endoscopy, we found the EREFS to accurately identify those with EoE. Children who responded to therapy had lower EREFS scores than non‐responders. EREFSs can be used to measure outcomes of pediatric patients, in conjunction with histology findings, and assess treatments for children with EoE.

Histamine drives severity of innate inflammation via histamine 4 receptor in murine experimental colitis

Ulcerative colitis (UC) patients exhibit elevated histamine, but how histamine exacerbates disease is unclear as targeting histamine 1 receptor (H1R) or H2R is clinically ineffective. We hypothesized that histamine functioned instead through the other colon-expressed histamine receptor, H4R. In humans, UC patient biopsies exhibited increased H4R RNA and protein expression over control tissue, and immunohistochemistry showed that H4R was in proximity to immunopathogenic myeloperoxidase-positive neutrophils. To characterize this association further, we employed both the oxazolone (Ox)- and dextran sulfate sodium (DSS)-induced experimental colitis mouse models and also found upregulated H4R expression. Mast cell (MC)-derived histamine and H4R drove experimental colitis, as H4R–/– mice had lower symptom scores, neutrophil-recruitment mediators (colonic interleukin-6 (IL-6), CXCL1, CXCL2), and mucosal neutrophil infiltration than wild-type (WT) mice, as did MC-deficient KitW-sh/W-sh mice reconstituted with histidine decarboxylase–deficient (HDC−/−) bone marrow–derived MCs compared with WT-reconstituted mice; adaptive responses remained intact. Furthermore, Rag2−/− × H4R−/− mice had reduced survival, exacerbated colitis, and increased bacterial translocation than Rag2−/− mice, revealing an innate protective antibacterial role for H4R. Taken together, colonic MC-derived histamine initiates granulocyte infiltration into the colonic mucosa through H4R, suggesting alternative therapeutic targets beyond adaptive immunity for UC.